The laboratory of Michael Yarmolinsky has continued its study of the bacterial equivalent of mitosis. In eukarotes, errors in mitosis are associated with developmental abnormalities and cancer. In prokaryotes the process has remained ill understood despite the apparent simplicity of the known components. To study the process the Yarmolinsky laboratory is making use of the simplest genetic elements that are equitably distributed to daughter cells at cell division, namely plasmids of low copy number such as prophage P1. Plasmid-encoded participants in this partitioning process are few: two proteins, ParA and ParB, and a DNA site, parS, the plasmid centromere. Interactions of wild type and mutant components of the partition apparatus have been studied in vivo by assays of (1) plasmid retention under non-selective conditions, (2) the expression of genes at various distances from parS, including reporter genes fused to genes of the par operon itself (3) immunoprecipitated DNA reversibly cross-linked to protein, (4) plasmid topoisomer distributions as displayed in one- and two-dimensional gels and (5) fractionation of cell components. During the period of this report, Dr. Yarmolinsky devoted his efforts towards completion of projects with which his laboratory has long been concerned. One study completed this year reevaluates provocative findings on the role in plasmid partitioning of a ParB polymerization that nucleates at parS and can spread along several kilobases of flanking DNA (Rodionov et al., 1999; Rodionov and Yarmolinsky, 2004). Other studies concern plasmid pairing and how ParA and plasmid replication influence the physical state of the plasmid. ParB was previously shown by an ingenious, albeit indirect, assay to be responsible for pairing plasmid centromeres, a presumed first step in the partitioning process (Edgar et al., 2001). This ParB-mediated pairing was detected in vivo by its effect in constraining DNA rotation in dimeric parS plasmids in appropriately treated bacterial constructs. Considerable effort has been spent this year on assessing how the presence of ParA influences plasmid pairing or otherwise anchors parS-bearing plasmids as reflected in the distribution of plasmid topoisomers. Much of this effort has gone into the construction and testing of bacterial strains suitable for assessing ParA effects on parS minicircles (DNA rings) devoid of an origin of replication and present in single copy per cell, i.e. capable of binding partition proteins, but incapable of replication or forming a plasmid pair. The study of ParA effects in this manner has turned out to be fraught with unexpected obstacles that have caused repeated delays. Although data on topoisomer distributions provide only an indirect indication of the molecular transactions being studied, the development of the parS minicircle as a tool with which to study partitioning in isolation from the act of replication (or even from recruitment to a site of replication) represents a substantial contribution. The work of the laboratory was largely carried out by two visiting scientists during this year, Drs. David Lane and Donald Biek. Dr. Lane developed the pairing assay for use with F plasmid and continued work initiated here on the selection of host mutants that affect the spreading of ParB protein as assayed by gene silencing. This work will be continued here and completed in Dr. Lane's laboratory in Toulouse. Dr. Biek is responsible for a major effort to develop the parS minicircle system and to solidify the evidence that ParA can anchor monomeric parS plasmids in the presence of ParB. Some of the experiments in a paper in press that clarifies the relationship between plasmid partition incompatibility and plasmid replication (Bouet et al., 2004), in which both Drs. Biek and Lane are co-authors, make use of strains and techniques developed in this laboratory. Finally, a project that has occupied Dr. Yarmolinsky for a period of several years, the analysis of the genome of bacteriophage P1, is now in press (Lobocka et al., 2004) accompanied by an invited guest commentary (Yarmolinsky, 2004). Two former post-doctoral fellows of this laboratory participated in the P1 genome project. In addition to supervising and participating in the research of the laboratory, Dr. Yarmolinsky has devoted considerable effort to reviewing manuscripts for major journals, reviewing grants, and otherwise participating in the scientific life of the scientific community at NIH and internationally. Work in progress was reported at international meetings in Sante Fe (Keystone) and Key Biscayne (ASM).ReferencesBouet, J.-Y., Rech, J., Egloff, S., Lane, D., and Biek, D.P. (2004) Probing plasmid partition with centromere-based incompatibility. Mol Microbiol (in press).Edgar, R., Chattoraj, D.K., and Yarmolinsky, M. (2001) Pairing of P1 plasmid partition sites by ParB. Mol Microbiol 42: 1363-1370.Lobocka, M.B., Rose, D.J., Plunkett, G., III, Rusin, M., Samojedny, A., Lehnherr, H., Yarmolinsky, M.B., and Blattner, F.R. (2004) The genome of bacteriophage P1. J Bacteriol.186 (in press).Rodionov, O., Lobocka, M., and Yarmolinsky, M. (1999) Silencing of genes flanking the P1 plasmid centromere. Science 283: 546-549.Rodionov, O., and Yarmolinsky, M. (2004) Plasmid partitioning and the spreading of partition protein ParB. Mol Microbiol 52: 1215-1223.Yarmolinsky, M.B. (2004) Bacteriophage P1 in retrospect and in prospect. J. Bacteriol. 186: (in press).